In recent years, the demand for an antibody drug having limited side effects, based on the antibody has been increasing yearly. Affinity chromatography using a carrier on which Protein A is immobilized is widely used as a means for purifying an antibody. Protein A as used here refers to Staphylococcus aureus-derived Protein A (described in A. Forsgren and J. Sjoquist, J. Immunol. (1966) 97, 822-827) or a portion of the domain sequences constituting the protein, capable of exhibiting the function of binding to an antibody.
A typical method for purifying an antibody using affinity chromatography using a carrier on which Protein A is immobilized is as follows. That is, the method involves (1) passing a raw material crude solution containing antibodies through a column filled with a carrier on which Protein A is immobilized to adsorb a desired antibody to the carrier, (2) washing the carrier to which the antibody is adsorbed with a neutral buffer solution to remove impurities, and then (3) eluting the antibody adsorbed to the carrier using an acidic buffer solution (specifically, pH 2.5 to 3.0).
The present inventors developed a technique involving introducing a Protein A-derived protein as a protein specifically binding to the Fc domain of an antibody while controlling orientation into an insoluble carrier by controlling orientation, as a means for efficiently purifying the antibody by increasing the capacity of binding to an antibody per carrier (see Patent Literatures 1 to 16).
The orientation-controlled immobilization method developed by the present inventors is as follows when expressed in a more generalized form.
The method is an orientation-controlled immobilization method comprising using a protein consisting of an amino acid sequence represented by the general formula: R1-R2-R3-R4-R5-R6, (wherein the sequence represents a sequence from the amino-terminal side towards the carboxyl-terminal side; the sequence of the R1 part may be absent, and, if present, is a sequence composed of amino acid residues other than lysine and cysteine residues; the sequence of the R2 part is a sequence of a Protein A-derived protein to be immobilized and a sequence adapted to comprise no lysine or cysteine residue so that it can hold a characteristic of binding to an antibody; the sequence of the R3 part may be absent, and, if present, is a spacer sequence composed of amino acid residues other than lysine and cysteine residues; the sequence of the R4 part is a sequence composed of 2 residues of amino acids represented by cysteine-X (wherein X represents lysine or an amino acid residue other than cysteine); the sequence of the R5 part may be absent, and, if present, is a sequence not comprising lysine or cysteine residues and a sequence comprising an acidic amino acid residue capable of rendering the isoelectric point of the whole protein consisting of an amino acid sequence represented by the general formula: R1-R2-R3-R4-R5-R6 on the acidic side; and the sequence of the R6 part is an affinity tag sequence for the purification of proteins) to cyanate the SH group of the sole existing cysteine residue in the R4 part, and reacting the cyanocysteinated protein with an insoluble carrier into which an amino group is introduced as a functional group to immobilize the part represented by R1-R2-R3 by the amide bonding between the carboxy terminus thereof and the amino group of the carrier.
Also in the purification of an antibody using the carrier immobilized by the orientation control of Protein A heretofore developed by the present inventors, a purification means has been applied which involves (1) passing a raw material crude solution containing antibodies through a column filled with a carrier on which Protein A is immobilized to adsorb a desired antibody to the carrier, (2) washing the carrier to which the antibody is adsorbed with a neutral buffer solution to remove impurities, and (3) eluting the antibody adsorbed to the carrier using an acidic buffer solution (specifically, pH 2.5 to 3.0), providing a means for efficiently purifying an antibody by the increased capacity of binding to an antibody per carrier.
However, the above method involving eluting an antibody using an acidic buffer solution having a pH of 2.5 to 3.0 has a serious flaw. Specifically, there is a problem that the contact of an antibody with a strongly acidic (e.g., pH 3.0 or less) buffer solution causes the acid modification of the antibody and changes the higher-order structure of the antibody, resulting in the induction of aggregation and the loss of the activity of the antibody as a medicine. The development of a more stable antibody molecule is attempted in order to solve the problem; however, it is obvious that such a solution cannot be a routine solution for a problem attending purification by affinity chromatography using a carrier on which Protein A is immobilized, and there is a need for the development of a method capable of being widely applied to the preparation of many antibody molecules.